The current method of choice for amplifying specific target DNA sequences is the Polymerase Chain Reaction (PCR) technique described generally in Mullis et al., U.S. Pat. No. 4,683,195. General features of PCR are shown schematically in FIG. 1. One begins with double-stranded DNA 10 containing a sequence of interest 12. The sequence of interest 12 is flanked by "primer target" sequences 14, 16. Primers 18, 20 are added to the DNA 10 along with a DNA polymerase and deoxyribonucleoside triphosphates. (Usually, a heat-stable DNA polymerase is employed to ensure that the polymerase activity is not destroyed by the heating required for denaturation.) The primers 18, 20 are single-stranded DNA oligonucleotides having sequences complementary to the primer target sequences 14, 16, respectively. The resulting mixture is heated to denature the DNA 10. After denaturation, the mixture is cooled sufficiently to allow the primers 18, 20 to anneal to the primer target sequences 14, 16, respectively, forming primed duplexes 21, 22, respectively. The primed duplexes 21, 22 are capable of being enzymatically extended. Since the polarity of each primer 18 is opposite the polarity of the other primer 20, replication of the sequence of interest 12, beginning from the 3' end of each primer 18, 20, will occur on both target strands 12a, 12b, respectively, of the sequence of interest 12. (In FIG. 1, the arrows 23, 24 denote the replication direction of primed duplexes 21 and 22.) During a "cycle" of replication, a strand complementary to each strand 12a, 12b of the sequence of interest is synthesized, wherein each strand 12a produces a complementary strand 12b (along with primer target 16) and each strand 12b produces a complementary strand 12a (along with primer target 14). After each cycle of replication, the reaction mixture is heated to denature the newly synthesized strands from their complementary parent strands. This cycle is repeated as many times as necessary to obtain the desired quantity of DNA of the sequence of interest 12. During each cycle of replication, primers anneal not only to the strands from the original sequence of interest, but also to strands produced by each round of replication. Thus, the number of copies of the sequence of interest 12 substantially doubles during each cycle. After multiple cycles, a large amount of the DNA from the sequence of interest 12 is produced that can be sequenced, cloned, or visualized on a gel.
Although PCR empowers users to amplify nucleic acid sequences exponentially, it has certain drawbacks. For example, replication from each primer must proceed in the direction of the primer on the complementary strand. Thus, only sequences located between primer target sequences can be amplified by PCR. However, it is often necessary or desirable to amplify sequences located outside a region flanked by primer target sequences.
Another disadvantage of PCR is that it requires two primers, thereby requiring that the practitioner have a detailed knowledge of sequences found in two separate regions near the sequence of interest. This information is not always available or readily obtainable.